The present disclosure relates generally to the field of DNA analysis. More particularly, the present disclosure relates to methods and systems for removing interferences from test samples, e.g., DNA-containing samples obtained from living subjects, when they are submitted for or subjected to molecular assays.
The copying and cloning of virtually any nucleic acid sequence has been greatly facilitated by the polymerase chain reaction (PCR), which has become a fundamental methodology in molecular biology. In its simplest form, the PCR is an in vitro method for the enzymatic synthesis of specific DNA sequences. In brief, the PCR involves hybridizing primers to the denatured strands of a target nucleic acid or template in the presence of a polymerase enzyme and nucleotides under appropriate reaction conditions. The polymerase enzyme (usually a thermostable DNA polymerase) then recognizes the primer hybridized to the template and processes a primer extension product complementary to the template. The resultant template and primer extension product can then be subjected to further rounds of subsequent denaturation, primer hybridization, and extension as many times as desired in order to increase (or amplify) the amount of nucleic acid which has the same sequence as the target nucleic acid. Commercial vendors market PCR reagents and publish PCR protocols. The PCR is capable of producing a selective enrichment of a specific DNA sequence by a factor of 109. The method is described in, e.g., U.S. Pat. Nos. 4,683,202; 4,683,195; 4,800,159; and 4,965,188, and in Saiki et al., 1985, Science 230:1350.
The optimal efficiency of the amplification reaction, however, may be compromised by a number of unwanted side reactions. For example, many PCR procedures yield non-specific by-products caused by mispriming of the primers and template. Primers hybridizing to each other may also result in lost efficiency. This problem may be particularly acute when the target nucleic acid is present in very low concentrations and may obscure any amplified target nucleic acid (i.e., may produce high background).
Also, masking agents which interfere or inhibit such molecular assays as the PCR are a problem in the art. Such inhibitors, which include leukocyte esterases, heme proteins, e.g., myoglobin and hemoglobin analogs, oxidation and breakdown products such as ferritins, methemoglobin, sulfhemoglobin and bilirubin, affect the accuracy of the assay, masking the true or detectable amount of, e.g., DNA in the sample. It is also conceivable that, e.g., a human sample containing genetic material for analysis could be spiked or doped with such agents to render a molecular assay done on the sample less trustworthy, or inconclusive.
Modem testing and treatment procedures have successfully reduced the prevalence and severity of many infectious diseases. For example, sexually-transmitted disease (STD) clinics regularly screen and treat patients for such diseases as gonorrhea and syphilis. Infectious agents such as gonococci may be identified by analyzing a DNA sample. Genetic transformation tests (GTT), such as the Gonostat® procedure (Sierra Diagnostics, Inc., Sonora, Calif.), can be used to detect gonococcal DNA in specimens taken from the urethra of men, and the cervix and anus of women. See, e.g., Jaffe et al., Diagnosis of gonorrhea using a genetic transformation test on mailed clinical specimens, J. Inf. Dis. 1982; 146:275-279, and Whittington et al., Evaluation of the genetic transformation test,. Abstr. Ann. Meeting. Am. Soc. Microbiol. 1983; p. 315. The Gonostat® assay is discussed in Zubrzycki et al., Laboratory diagnosis of gonorrhea by a simple transformation test with a temperature-sensitive mutant of Neisseria gonorrhoeae, Sex. Transm. Dis. 1980; 7:183-187. The Gonostat(3) GTT, for example, may be used to detect, e.g., gonococcal DNA in urine specimens. The Gonostat assay uses a test strain, N. Gonorrhoeae, ATCC 31953, which is a mutant strain that is unable to grow into visible colonies on chocolate agar at 37° C. in 5% CO2. Gonococcal DNA extracted from clinical material can restore colony growth ability to this test strain.
Such tests such can be used to detect DNA in such bodily fluids and excretions as urine, blood, blood serum, amniotic fluid, spinal fluid, conjunctival fluid, salivary fluid, vaginal fluid, stool, seminal fluid, and sweat. Another test that can be used to identify DNA in a bodily fluid is the PCR, since it uses discrete nucleic acid sequences and therefore can be effective even in the absence of intact DNA.